Mechanism of regulation of vitamin D metabolism at molecular level.

分子水平上维生素 D 代谢的调节机制。

• 批准号: 08457494
• 负责人: SHINKI Toshimasa
• 金额: $ 4.93万
• 依托单位: Showa University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08457494/
• 关键词: parathyroid hormone; 1alpha, 25-dihydroxyvitamin D_3; 1alpha-hydroxylase; 24-hydroxylase; proximal convoluted tubules; cytochrome P450; vitamin D receptors; degenerate-PCR; 近位尿細管; ネフロン; 腎臓

The vitamin D endocrine system is regulated reciprocally by renal 25-hydroxyvitamin D_3 1alpha- and 24-hydroxylases, Previously, we reported that renal proximal convoluted tubules, the major site of 1alpha, 25-hydroxyvitamin D_3 production, have vitamin D receptors. In the presence of vitamin D receptors, renal proximal convoluted tubules cannot maintain the state of enhanced production of 1alpha, 25-dihydroxyvitamin D_3. To clarify this discrepancy, we proposed a working hypothesis for the reciprocal control of renal 25-hydroxyvitamin D_3 1alpha- and 24-hydroxylase activities. In rat models of renal production of 1alpha, 25-hydroxyvitamin D_3, expression of vitamin D receptors and 25-hydroxyvitamin D_3 24-hydroxylase mRNAs was strikingly suppressed in renal proximal convoluted tubules but not in the cortical collecting ducts.By using the method of mixed oligonucleotide-primed amplification of cDNA,we have cloned a new sequence of the cytochrome P450 mixed function oxidase from the kid … More ney of vitamin D-deficient rats. A full-length cDNA for the rat kidney mitochondrial cytochrome P450 mixed function oxidase, 25-hydroxyvitamin D_3-1alpha-hydroxylase (P4501alpha), was cloned from a vitamin D-deficient rat kidney cDNA library and subcloned into the mammalian expression vector pcDNA 3.1 (+). When P4501alpha cDNA was transfected into COS-7 transformed monkey kidney cells, they expressed 25-hydroxyvitamin D_3-1alpha-hydroxylase activity. The sequence analysis showed that P4501alpha consisted of 2469bp in length and contained an open reading frame encoding 501 amino acids. The deduced amino acid sequence showed less than 30% similarity to those of all other P450s reported to date. Thus, it is a new member of the P450 superfamily. The expression of P4501alpha mRNA was greatly increased in the kidney of vitamin D-deficient rats. In rats with the enhanced renal production of 1alpha, 25-dehydroxyvitamin D_3 (rats fed a low Ca diet), expression of P4501alpha mRNA was greatly enhanced in the renal proximal convoluted tubules. These results clearly demonstrate that the expression of P4501alpha is regulated at a transcriptional level. Less

维生素D内分泌系统是由肾脏25-羟维生素D_3 1 α和24-羟化酶相互调节的。以前，我们报道过肾近曲小管是产生25-羟维生素D_3的主要部位，它含有维生素D受体。在维生素D受体存在的情况下，肾近曲小管不能维持增强生产1α， 25-二羟基维生素D_3的状态。为了澄清这种差异，我们提出了肾脏25-羟基维生素d_31 α -和24-羟化酶活性相互控制的工作假设。在肾生成1 α， 25-羟基维生素D_3的大鼠模型中，维生素D受体和25-羟基维生素D_3 24-羟化酶mrna的表达在肾近曲小管中明显抑制，而在皮质集管中没有。利用混合寡核苷酸引物扩增cDNA的方法，从维生素d缺乏症大鼠体内克隆了一个新的细胞色素P450混合功能氧化酶序列。从维生素d缺乏大鼠肾脏cDNA文库中克隆大鼠肾脏线粒体细胞色素P450混合功能氧化酶25-羟基维生素d_3 -1 α -羟化酶（p4501α）全长cDNA，并将其亚克隆到哺乳动物表达载体pcDNA 3.1（+）中。将P4501alpha cDNA转染到COS-7转化的猴肾细胞中，它们表达了25-羟基维生素d_3 -1 α -羟化酶活性。序列分析显示，P4501alpha全长2469bp，包含一个编码501个氨基酸的开放阅读框。推导出的氨基酸序列与迄今为止报道的所有其他p450的相似性小于30%。因此，它是P450超家族的新成员。p4501α mRNA在维生素d缺乏大鼠肾脏中的表达显著升高。在肾脏生成1α， 25-去羟基维生素D_3增强的大鼠（低钙饮食的大鼠）中，肾近曲小管中P4501alpha mRNA的表达大大增强。这些结果清楚地表明P4501alpha的表达在转录水平上受到调控。少

项目成果

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